The complement system is an important means by which a host defends itself against infection. The complement cascade system is a component of the immune system that helps provide a natural immunity against invading microbes and is also an effector arm of antibody mediated humoral immunity. Complement is responsible for activating cells and other molecules involved in the inflammatory process as well as being directly related to the destruction of microbial invaders. The activation of complement involves a cascade of proteolytic reactions that lead to the release of inflammatory mediators and result in the assembly of the microbial membrane attack complexes which, in turn, lyse invading microbial cells. This cascade system has been characterized as containing at least thirty serum and membrane proteins that are activated by antibody-antigen complexes or by the invasion, in a host or experimentally in culture, by a microorganism, or other antigenic molecules. Complement proteins may be grouped into three general categories: activating components, receptors, and positive and negative regulators.
The complement cascade consists of two major branches, the classical and alternative pathways. Though these pathways are initiated differently, they converge at the step of complement protein C3 activation (see FIG. 5). The complement cascade can mediate undesirable cellular damage in inflammatory, immune or autoimmune (auto-antibody-mediated) conditions such as; myasthenia gravis, immune complex excess syndromes such as systemic lupus erythematosus, ischemia-reperfusion states, hyper-acute rejection of transplants, organ failure conditions such as adult respiratory distress syndrome, Alzheimer's disease and related neurodegenerative disorders, among others.
A series of regulatory proteins are involved in the control of the complement cascade. These proteins are considered part of the complement system and act to block endogenous complement activity at either initiation or the formation of the membrane attack complex. various therapeutic agents are being developed that block different steps in the complement cascade.
Complement is a group of serum proenzymes that are activated by antigen bound immunoglobulin or by membrane components on gram negative bacteria or fungi. The alternative pathway of the complement system is initialized by either the introduction of an endotoxin such as lipopolysaccharide [LPS], a component of the cell walls of gram negative bacteria or for instance by zymosan, a component of yeast cell walls, or by aggregated IgA.
The classical pathway of the complement system is initialized by Complement protein C1 binding to antigen bound IgG or IgM. Both pathways converge at the formation of C3 convertase at which point an amplification takes place that generates literally thousands of C3a and C3b fragments. C3b fragments can bind to complement protein complex C4b2a to form C4b2a C3b which is called C5 convertase and generates thousands of C5a and C5b fragments. C3b can also be used to regenerate C3 convertase which causes a greater amplification of complement protein split product C3a. Split products C3a and C5a interact with receptors on mast cells to cause them to release histamine. Histamine induces inflammation which is generally considered protective, but in conditions characterized by improper complement activation and/or regulation inflammation can lead to damaged tissue.
One approach to inhibit complement mediated effects is by depleting complement. Depleting complement involves reducing the proteins responsible for the regeneration of C3 or C5 convertase and thereby reducing the amount of C3a and C5a produced. In this way complement is depleted or "used up". One such method for depleting complement component C3 convertase involves allowing C3 convertase to form and then binding split product C3b in order to reduce the further amplification of C3 convertase formation which can lead to C5 convertase formation.
In another approach for inhibiting complement the pathway is inhibited before the formation of C3 convertase. Inhibition of the formation of C3 convertase limits the production of split products C3b and C3a and further limits the formation of C5 convertase. Using this approach complement activation is blocked rather than depleted.
Candinas et al., describe the activation and depletion of complement by using cobra venom factor in conjunction with a recombinant soluble complement receptor type 1 protein (sCR1), and the use of such molecules in treating hyperacute xenograft rejection. (Candinas, D. et al., Transplantation 1996 15;62(3):336-342) sCR1 is a recombinant protein that has been shown to inhibit both the classical and alternative pathways of complement and thereby limits the production of proinflammatory products such as the anaphylatoxins (complement proteins C3a, C4a and C5a). sCR1 has also been described by Moore, F D Jr., as the first protein useful to treat adverse clinical situations which are complement-dependent, and further describes potential uses for sCR1 to treat thermal injury, ARDS, septic shock, and ischaemia/reperfusion injury events such as myocardial infarction after thrombolytic therapy. (Moore, F D Jr., Adv. Immunol 1994 56:267-299) U.S. Pat. No. 5,856,297, Fearon et al., issued Jan. 5, 1999 claims pharmaceutical compositions comprising a CR1 protein in various modifications and describes CR1 and the recombinant forms of the protein as being useful in the diagnosis and treatment of disorders involving complement activity and inflammation.
Other proteins have been investigated for their usefulness in inhibiting or modulating complement. For instance, Human IgG has been used to balance complement activation in a pig-to-primate cardiac xenotransplantation hyperacute rejection study. The study determined that Human IgG caused a dose-dependent decrease in deposition of complement protein iC3b and a decrease in formation of C3 convertase. Furthermore, the infusion of IgG was found to prevent hyperacute rejection of porcine hearts transplanted into the primates. Magee J. C. et al., J.Clin.Invest 1995 96(5):2404-2412. U.S. Pat. No. 5,851,528, Ko et al., issued Dec. 22, 1998, U.S. Pat. No. 5,679,546, Ko et al., issued Oct. 21, 1997, and U.S. Pat. No. 5,627,264, Fodor et al., issued May 6, 1997, describe chimeric proteins useful in inhibiting complement activation and describe methods to treat adverse conditions related to complement mediated inflammation. Sims, et al., U.S. Pat. No. 5,550,508, issued Aug. 27, 1996, describes polypeptides which act to inhibit complement C5b-9 complex activity. The protein is an 18 kDA protein found on the surface of human erythrocytes and is described as being useful in treating immune disease states when administered in effective amounts.
Magee, J. C. et al., J. Clin. Invest. 1995 96(5):2404-12, investigated the use of immunoglobulin to prevent complement-mediated hyperacute rejection in swine-to-primate xenotransplantation. In the study human IgG was added to human serum and was found to cause a dose-dependent decrease in the deposition of iC3b, cytotoxicity, and heparin sulfate release when the serum was incubated with porcine endothelial cells. It appears as if the decrease was caused by a decrease in the formation of C3 convertase on the endothelial cells. Furthermore, infusion of purified human IgG into primates prevented hyperacute rejection of porcine hearts in a xenotransplantation. Magee et al., determined that such results support the use of IgG as a therapeutic agent in humoral-mediated disease conditions.
U.S. Pat. No. 4,374,831, Joseph et al., issued Feb. 22, 1983, U.S. Pat. No. 4,087,548, Lenhard et al., issued May 2, 1978, U.S. Pat. No. 4,021,545, Nair et al., issued May 3, 1977, and U.S. Pat. No. 3,998,957, Conrow et al., issued Dec. 21, 1976, all describe chemical molecules which are useful as inhibitors and/or modulators of complement. Josephe et al., describe Bis-(.beta.-D-glucopyranosyl-1-oxy)-arylene sulfate derivatives and methods for modulating complement in a warm blooded animal using pharmaceutical compounds comprising such molecules. Lenhard et al., describe complement inhibitory compounds such as C-substituted trisulfonic acids, acid ureides, and oxalyl amides and methods for inhibiting the complement system in a warm blooded animal by administering complement inhibitory amounts of the compounds comprising such molecules. Nair et al., claim methods for inhibiting the complement system in a warm blooded animal by using compositions comprising Inulin poly(H-sulfate). Conrow et al., describe 1,1'-[ureylenebis(sulfo-p-phenylene)]bis{sulfo-1H,8H-indazolo{2,3,4-cde]be nzotriazol-9-ium hydroxide}, bis(inner salts), and tetra salts as useful complement inhibitors.
In WO 95/32719 (1995), Galbraith describes the use of phosphorothioate oligonucleotides for depleting complement. The approach described involves administering, to a primate, an oligonucleotide 2 to 50 nucleotides in length containing at least one phosphorothioate internucleotide linkage, thereby stimulating vasodilation, and reducing complement activity by depleting complement.
Lin et al., WO 97/42317, describe oligonucleotides (aptamers) having phosphorothioate and/or substituted phosphonate linkages that are 37-61 base pairs in length which bind complement protein C3b and may be used diagnostically in vivo or in vitro to detect C3b in a biological sample. The aptamers may be used therapeutically to inhibit undesirable C3b-mediated complement events such as inflammation.
There has been and continues to be a long-felt need for methods for the inhibition and/or modulation of the complement mediated immune response using modified oligonucleotide compounds that might incorporate modifications for improving characteristics such as compound stability and cellular uptake. Such methods would be useful for therapeutically and prophylactically, as well as for diagnostic reagents and research reagents including reagents for the study of both cellular and in vitro events.